This project will exploit a recently discovered oncogenic mechanism of cell cycle control exerted on cyclin D- cdk4 (DK4) by p27Kip1 and its activator, the Breast tumor Related Kinase (Brk), to target vulnerabilities in cancer cells. Although historically known as a DK4 assembly factor and cdk2 inhibitor, p27 also acts as a cdk4 ON/OFF ?switch.? Tyrosine (Y) phosphorylation of p27 (pY88) gatekeeps both ATP binding and CAK phosphorylation of cdk4's T loop, essential for DK4 activation. We demonstrated that Brk is the physiological kinase that phosphorylates p27, and by doing so, increases cdk4 activity, cell cycle progression, and resistance to cdk4-specific inhibition by the chemical inhibitor Palbociclib, currently in clinical trials for breast cancer. Cyclin D and cdk4 are overexpressed in tumors, but their levels are not reliable biomarkers of oncogenic tumor potential because of the activating role p27 has on their complex. Our data predict that the Brk-p27-DK4 axis is important for transformation in DK4-dependent tumors and can explain tumor resistance to cdk4-specific inhibition. We determined that p27 interacts with Brk through a SH3:PxxP contact, specific for Brk's SH3 domain. The importance of this interaction is validated by characterization of a natural ALTernatively spliced Brk variant Brk (ALT), which contains only Brk's SH3 domain, and functions as a competitive inhibitor in vivo. Thus, blocking the Brk:p27 interaction is a viable strategy to inhibit cdk4 activity, which should be explored therapeutically. Our specific aims: 1) To examine pY as a marker of Palbociclib response in tissue culture and patient material. We will characterize cultured cell lines to statistically establish whether pY, as a measure of cdk4, correlates with Palbociclib sensitivity and could be used to predict response. In conjunction with our Dept. of Pathology, we will analyze archival breast cancer samples and primary patient material, including ER/PR+, Her- breast tumors, to correlate Palbociclib responsiveness and pY directly. 2) To demonstrate that ALT and pY blockage therapy can prevent proliferation in tissue culture models. Using breast cancer tissue culture models, we will show that ALT and Palbociclib-mediated arrest is different in terms of outcome and at the molecular and cell biological level. Our data will show that blocking pY using ALT is a powerful approach to block cancer cell proliferation, because it inhibits both cdk4 and cdk2, induces cellular senescence and prevents drug resistance. 3) To demonstrate that ALT and pY blockage therapy can prevent tumor progression in animal models. We will show that blocking p27 pY in mouse xenografts breast cancer models can prevent cancer progression and drug resistance.